Source: UNIVERSITY OF CALIFORNIA, DAVIS submitted to NRP
THE MOLECULAR AND CELLULAR BIOLOGY OF GAMETE INTERACTIONS INFERTILIZATION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0157986
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2001
Project End Date
Sep 30, 2006
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
ANIMAL SCIENCE
Non Technical Summary
The objective of this project is to increase the knowledge of reproductive biology relevant to agriculturally important animals, knowledge that will provide the understanding needed to regulate reproductive efficiency. Research will focus on gamete (eggs and sperm) biology and the fertilization process. Animal systems used will include fish, frogs, and mammals. Models systems will be used e.g., frogs, where molecular and cellular experimental approaches will likely be more successful. Findings from these model systems will be tested in agriculturally important organisms such as pigs and fish. The methods used in this project will include contemporary methods of proteomics, functional genomics, and structural biology as well as more classical methods of biochemistry and cell biology.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3013510100010%
3013712100010%
3013711100010%
3013799100030%
3013799103010%
3013799104030%
Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3711 - Trout; 3510 - Swine, live animal; 3712 - Salmon; 3799 - Cultured aquatic animals, general/other;

Field Of Science
1040 - Molecular biology; 1030 - Cellular biology; 1000 - Biochemistry and biophysics;
Goals / Objectives
Optimizing reproductive performance in agriculturally important animals, including aquaculture species is of major importance for efficient animal production. The long term goal of this project is to increase our knowledge of reproductive biology processes related to gamete biology and fertilization. The project will provide a molecular understanding of the glycoprotein composition, structure, and biological role of the egg extracellular matrix in fertilization and early development. Specific objectives are: 1) Complete studies on determining the number of glycoprotein constituents in egg envelopes and cDNA cloning of these molecules. This objective will use proteomic methods (1D- and 2D-electrophoresis for separation of glycoproteins, determination of mass and amino acid sequence) and molecular biology methods (clone those molecules not currently in the protein and nucleic acid database). This objective will initially using the frog system and secondarily mammalian systems (pig and mouse). 2) Determine the mechanism of action of the ovary furin convertase on envelope glycoproteins synthesized by the liver and on the ovarian function of the protease. This objective will initially use a fish system and secondarily a frog system. 3) Identify the location of proteases in the egg (extracellular and cortical granule) and purify, characterize, and clone additional egg proteases that modify the egg extracellular matrix during the cortical reaction. Additional proteases will likely be identified using proteomic approaches to defining the composition of the cortical granule components in frog and fish eggs. Those cortical granule components not in the sequence database will be cloned and investigated as to their biochemical and biological actions on the egg extracellular matrix (functional genomics). This objective will use both frog and fish systems initially and secondarily mammalian systems. 4) Complete the cloning and characterization of the egg cortical granule glucosaminidase and determine the oligosaccharide structure of its extracellular matrix glycoprotein substrate, ZPC. This objective will initially use the frog system and secondarily mammalian systems. 5) Identification and structural characterization of the ligand for the cortical granule lectin. For the carbohydrate moiety of the ligand, mass spectrometry studies will be used to determine its structure. For the protein moiety, protein chemistry and molecular biology methods will be used. This objective will initially utilize frog and fish systems and secondarily mammalian systems (the pig).
Project Methods
The research strategy for this project will focus on gamete biology and fertilization using model animal systems where appropriate. The experimental approach will employ proteomic, bioinformatic, functional genomic, and structural biology methods. Objective 1) Proteomics methods will be used to identify new envelope glycoproteins from frog eggs. Gel electrophoresis (1D and 2D) will be used for separation of the glycoproteins and deglycosylated proteins. Peptides from the separated proteins will be produced 'in gel' and their masses and amino acid sequences determined by mass spectrometry and Edmann sequencing methods. From the amino acid sequences, nucleic acid primers will be made and using PCR and standard cDNA cloning methods with ovarian cDNA libraries, cDNAs for the glycoproteins will be cloned. Using the translated cDNA sequences, databases will be searched using FASTA and BLAST programs to identify related sequences and thereby infer function from structure conservation. Objective 2) Methods to be used in research on the fish ovary furin protease include immunocytochemical methods to localize the enzyme within oocytes and in the egg envelope. Expression of the enzyme in transformed cells (insect cells and the Baculovirus expression system) to obtain sufficient amounts of active enzyme and demonstrate the enzyme's specific proteolysis of envelope glycoproteins. The genomic DNA sequence of the furin protease will be determined using DNA sequencing methods to determine the enzyme's cell and tissue specific gene expression. Objective 3) The cellular location of frog egg proteases will utilize immunocytochemical methods. Additional egg proteases will be identified using proteomic methods (as in Objective 1) and database searching methods. A global approach to identifying the protein and glycoprotein composition of egg cortical granules (frog and fish) will use proteomic methods (2D-PAGE and mass spectrometry). Functional genomics methods will be used to investigate cortical granule components not in the protein and nucleic acid databases (cloning of molecules from proteomics derived amino acid sequence data using PCR, cDNA libraries, and standard cDNA cloning methods; expression of cloned cDNA and demonstration of the biological activities of expressed proteins/glycoproteins on the egg envelope). Objective 4) The frog egg glucosaminidase enzyme will be cloned using PCR primers derived from amino acid sequence data and standard cDNA cloning methods. The ZPC oligosaccharide structure of its substrate will be determined using chemical and mass spectrometry methods. Glycosidase action of the cloned and expressed glucosaminidase enzyme will be determined using mass spectrometry and HPLC methods. Objective 5) Isolation of the ligand for the cortical granule lectin will use biochemical methods (column chromatographic methods). The ligand will be isolated from frog, fish and pig eggs. Protein chemistry and molecular biology methods will be used to determine the structure of the protein moiety of the ligand. The structure of the oligosaccharide moiety of the ligand will be determined using mass spectrometry.

Progress 10/01/01 to 09/30/06

Outputs
Due to a lack of research funds, progress during the last year was minimal with no publications. Research projects under investigation included: 1) computer modeling and binding studies on the oviductal enzyme oviductin, 2) determination of the disulfide bond linkages in the Chinook salmon egg lectin, and 3) determination of the oligosaccharide structures of the envelope glycoprotein ZPC from Xenopus laevis. I anticipate that these projects will be concluded during the coming year with one publication from each project.

Impacts
The impact of these projects will be on our basic understanding as to the cell biology of sperm-egg interactions and the molecular mechanisms of the fertilization process. This understanding will be potentially useful in controlling the reproductive process at the level of fertilization.

Publications

  • No publications reported this period


Progress 01/01/05 to 12/31/05

Outputs
1) The protease responsible for ZPA hydrolysis in Xenopus laevis was purified from activated egg exudate to near homogeneity using ion exchange chromatography. Inhibitor studies indicated that the protease is a zinc metalloprotease sensitive to 1,10-phenanthroline, EDTA, and hydroxamate inhibitors. Protease treatment of egg envelopes resulted in envelope hardening, and a peptide-specific hydroxamate inhibitor blocked this effect. Using antibodies specific for the cleaved N-terminal fragment of ZPA it was determined that the fragment remains tethered to ZPA following cleavage via a disulfide bond. These results indicate that ZPA proteolysis by an egg metalloprotease triggers a conformational change which hardens the envelope. 2) We investigated the antibody response and the effect on the estrous cycle following a single inoculation of porcine zonae pellucidae (pZP) employing controlled-release methodology in equids. Twenty-seven domestic mares were inoculated with various formulations of pZP and adjuvant. The anti-pZP antibodies persisted for at least 43 weeks (length of the study). Of the various formulations used in the study, pZP and QS-21 water-soluble adjuvant, administered in combination with an emulsified preparation of pZP and Freund's Complete Adjuvant generated a significantly (P < 0.05) higher titer of anti-pZP antibodies when compared with other formulations employing the water-soluble adjuvant, Carbopol. Hormone analyses indicated a high incidence and extended duration of persistent corpora lutea among the treated mares. The positive control group of mares also exhibited high incidences of persistent corpora lutea. However, all mares eventually returned to normal cyclicity. The basis for the high incidence and extended duration of persistent corpora lutea was unexplained. The results demonstrate for the first time the persistent generation of anti-pZP antibodies following a single inoculation of pZP incorporated into a controlled-released preparation in the horse. This study further suggests that a single inoculation of pZP sequestered in a controlled-release lactide-glycolide polymer may serve as an alternative to traditional two-inoculation protocols for contraception investigations in the equine.

Impacts
In mammals and frogs a prominent feature of envelope modification is the proteolysis of the egg envelope glycoprotein ZPA. We have identified the protease responsible for this process. We have improved the methodology for immunocontraception in wild and domestic animals using the pig zona pellucida.

Publications

  • Lindsay, L.L., and J.L. Hedrick. 2004. Proteolysis of ZPA Triggers Egg Envelope Hardening in Xenopus laevis. Biochem. Biophys. Res. Comm., 324, 648-654.
  • Liu, I.K.M., J.W. Turner, E.M.G. VanLeeuwen, D.R. Flanagan, J.L. Hedrick, K. Murata, J.F. Kirkpatrick, V.M. Lane, and M.P. Morales-Levy. 2005. Persistence of anti-zona pellucidae antibodies following a single inoculation of porcine zona pellucidae in the domestic equine. Reproduction, 129, 181-190.


Progress 01/01/04 to 12/31/04

Outputs
1) A Xenopus laevis egg cortical granule, calcium-dependent, galactosyl-specific lectin participates in forming the fertilization layer of the egg envelope and functions in establishing a block to polyspermy. We reported the cDNA cloning of the lectin, expression of the cortical granule lectin gene during oogenesis and early development, and identification of a new family of lectins. The translated cDNA for the cortical granule lectin had a signal peptide, a structural sequence of 298 amino acids, a molecular weight of 32.7 K, contained consensus sequence sites for N-glycosylation and a fibrinogen domain. The lectin cDNA was expressed during early stages of oogenesis and glycoprotein levels were constant during development. Lectin mRNA levels were from 100- to 1000-fold greater in ovary than in other adult tissues. The lectin had no sequence homology to the previously identified lectin families but had 41 to 88% amino acid identity with nine translated cDNA sequences from an ascidian, lamprey, frog, mouse, and human. Based on the conserved properties of these glycoproteins, we proposed a new family of lectins, the eglectin family. 2) A strategy combining accurate mass determination, tandem mass spectrometry (MS), structure homology, and exoglycosidases was reported for the structural characterization of mucin-type O-linked oligosaccharides. The method was used to profile with quantitation the O-linked oligosaccharide components of the only diploid Xenopus frog, Xenopus tropicalis. Collision-induced dissociation in MS was used to determine connectivity, to identify previously characterized oligosaccharides, and to determine the presence of structural motifs in unknown oligosaccharides. Exoglycosidase digestion was used to identify the individual residues along with the linkages. The enzymes were also used to cleave larger oligosaccharides to smaller units that are similar to previously elucidated components. A total of 35 oligosaccharides including neutral, sialylated, and sulfated, were identified. The relative abundances of all components were also determined based on HPLC. 3) The morphological distribution of oligosaccharides was determined in the three Xenopus laevis egg jelly layers. The jelly coat contains three morphologically distinct layers designated J1, J2, and J3 from the innermost to the outermost and is composed of 9 to 11 distinct glycoproteins. Each jelly layer is known to have specific functions in the fertilization of the egg. Oligosaccharide identifications were based on HPLC retention times (porous graphitized carbon column), exact masses, and tandem mass spectrometry. Over 40 neutral and 30 sulfated oligosaccharides were observed in the three jelly layers. Neutral oligosaccharide structures from different jelly layers were both unique and overlapping, while sulfated oligosaccharides were detected only in layers J1 and J2. Neutral oligosaccharides unique to jelly layer J3 and the combined layers J1/J2 had similar core structures and similar residues, with differences between these two sets of unique oligosaccharides primarily due to the branching carbohydrate moieties.

Impacts
Studies of egg lectin established its role in blocking polyspermy at fertilization and led to the discovery of a 12th class of lectins, the eglectins. Eglectins likely play essential roles in biological processes in addition to a block to polyspermy at fertilization. Methods for oligosaccharide structure determination developed using Xenopus eggs will likely prove useful for studying glycoproteins in many biological systems.

Publications

  • Chang, Y. B., T. R. Peavy, N. J. Wardrip, and J. L. Hedrick. The Xenopus laevis cortical granule lectin: cDNA cloning, developmental expression, and a human homolog. Comparative Biochemistry and Physiology, 137, 115-129 (2004).
  • Zhang, J., L.L. Lindsay, J.L. Hedrick, and C.B. Lebrilla. A strategy for profiling and structural elucidation of mucin-type oligosaccharides by mass spectrometry. Anal. Chem., 76, 5990-6001 (2004).
  • Zhang, J., Y. Xie, J.L. Hedrick, and C.B. Lebrilla. Profiling the morphological distribution of O-linked oligosaccharides. Anal. Biochem., 334, 20-35, (2004).


Progress 01/01/03 to 12/31/03

Outputs
1) Xenopus laevis is a widely used vertebrate model system. It is not optimal for genetic manipulations due to its tetraploid genome. Xenopus tropicalis, has the advantages of a diploid genome and a shorter generation time. A comparative investigation of X. tropicalis egg extracellular matrix glycoproteins in relation to those already characterized in X. laevis was undertaken. Fertilization methods and isolation of egg extracellular molecules were directly transferable from X. laevis to X. tropicalis. Cross-fertilizations were successful in both directions. Almost identical SDS-PAGE gel patterns of envelopes was obtained, whereas jelly component profiles were similar only for the larger macromolecules (90 kDa). The X. tropicalis cDNA was cloned for egg envelope glycoproteins ZPA, AX, B, C, D and the cortical granule lectin; the X. tropicalis sequences were 85% identical to X. laevis. Thus, homologous molecules undoubtedly had the same functions. The molecular and cellular mechanisms of fertilization in these two species are probably equivalent. 2) A X. laevis egg cortical granule lectin (CGL) participates in forming the fertilization layer of the egg envelope and functions in establishing a block to polyspermy. We undertook the cDNA cloning of CGL, determined the expression of the CGL gene during oogenesis and early development, and identified of a new family of lectins. CGL cDNA was expressed during early stages of oogenesis and was constant during development. CGL mRNA levels were 100-1000 fold greater in ovary than in other adult tissues. CGL had no sequence homology to the twelve previously identified lectin families but had 41 to 88% amino acid identity with seven translated cDNA sequences from an ascidian, frogs, lamprey, mouse, and human. Based on the conserved carbohydrate binding and structural properties of these glycoproteins, we proposed a new family of lectins, the eglectin family. 3) We developed a method to determine glycosylation sites and oligosaccharide heterogeneity in glycoproteins based using non-specific proteolysis, deglycosylation, and matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS). Glycoproteins were digested with pronase to glycopeptides and amino acids. Non- glycosylated peptide fragments were digested to amino acids. Steric hindrance prevented hydrolysis of the peptide moieties attached to the glycans. Glycopeptides were desalted and concentrated using solid phase extraction and analyzed by MALDI-MS. The glycans were also analyzed by MALDI-MS after releasing the glycans from glycoproteins using PNGase F. The peptide moieties of the glycopeptides were identified by subtracting the masses of the glycans derived from PNGase F treatment from the masses of the glycopeptides. The experimental strategy was validated using glycoproteins with known oligosaccharide structures, Ribonuclease B and chicken ovalbumin. We determines the N-glycosylation sites and site heterogeneity of a glycoprotein the X. laevis egg CGL. This procedure is useful for determining protein site heterogeneity and structural heterogeneities of the oligosaccharide moieties of glycoproteins.

Impacts
The fertilization research previously done with X. laevis can be extrapolated to X. tropicalis. This will benefit future research as the X. tropicalis genome is being determined. Studies of CGL established its role in blocking polyspermy at fertilization and led to the discovery of a 12th class of lectins. Lectins play may essential roles in biological processes.

Publications

  • An, H. J., T.R. Peavy, J.L. Hedrick, and C.B. Lebrilla. 2003. Rapid Method for the Determination of N-glycosylation Sites and Site Heterogeneity. Anal. Chem., 75, 5628 - 5637.
  • Chang, Y.B., T.R. Peavy, N.J. Wardrip, and J.L. Hedrick. 2003. The Xenopus laevis cortical granule lectin: cDNA cloning, developmental expression, and a human homolog. Comparative Biochemistry and Physiology, 137, 115-129.
  • Lindsay, L.L, T.R. Peavy, R.S. Lejano, J.L. Hedrick. 2003. Comparison of Xenopus laevis and Xenopus tropicalis molecules involved in fertilization. Comparative Biochemistry and Physiology, Part A, 136, 343-352.


Progress 01/01/02 to 12/31/02

Outputs
1) A method with high speed and sensitivity was published for the analysis of O-linked oligosaccharides from the egg jelly coat of Xenopus laevis. The method relied primarily on matrix-assisted laser desorption-ionization Fourier-transform mass spectrometry (MALDI-FTMS) and collision-induced dissociation (CID). Partial structures of anionic components, composed primarily of sulfate esters, were obtained with CID. For neutral species, complete structures were obtained using a comparative method. In this method, a small number of oligosaccharide structures, elucidated previously by NMR, were used to develop a set of substructural motifs that were characterized by CID. The presence of the motifs in the CID spectra of jelly coat oligosaccharides were then used to determine the structures of unknown compounds that were in abundances too small for NMR analysis. 2) A study was published on the glycoprotein composition of vitelline envelopes (VEs) of Bufo arenarum eggs and their role in fertilization. The B. arenarum VE was biochemically similar to other egg envelopes. The biological properties of the VE was similar to other species in regard to sperm binding and induction of the acrosome reaction. However, as far as we are aware, this is the first observation of the VE inducing the sperm acrosome reaction in amphibians. The relatively small differences observed in heterologous sperm-VE binding (X. laevis and B. arenarum) are inconsistent with the current paradigm that species specificity in fertilization is regulated at the sperm-VE binding step. 3) We reported identification of a new X. laevis egg envelope glycoprotein. A cDNA sequence was obtained that represented a mature protein of 32 kDa. A BLAST analysis showed that it was related to the ZP domains of mammalian tectorin, uromodulin and ZPA. The new glycoprotein was unique among egg envelope glycoproteins and was designated ZPD. A similar glycoprotein was identified by immunocrossreactivity in Xenopus tropicalis and Xenopus borealis egg envelopes. 4) To understand the molecular mechanism of sperm-egg envelope binding, we characterized the N-linked glycans of all the X. laevis vitelline envelope glycoproteins. The N-linked glycans of the VE were composed predominantly of a heterogeneous mixture of high mannose and neutral, complex oligosaccharides primarily derived from ZPC. The majority of the ZPC N-linked complex oligosaccharides consisted of structurally related neutral glycans with terminal beta-N-acetyl-galactosamine, alpha-galactose, and/or alpha-fucose residues. The high mannose ZPC oligosaccharides were oligo-mannose 9 through 5 structures. One of the complex oligosaccharides of ZPC possessed terminal beta-N-acetyl-glucosamine residues that was not present in ZPC isolated from the activated egg envelopes. We previously showed that the cortical granules contain beta-N-acetyl-glucosaminidase. We propose that an alteration in the oligosaccharide structure of ZPC by glucosaminidase released from the cortical granule reaction is responsible for the loss of sperm binding ligand activity at fertilization.

Impacts
We improved methods to determine the structure of oligosaccharides and their role in fertilization and early development. Knowledge of egg envelope glycoprotein structure-function is useful in understanding sperm-egg interactions and the process of embryo hatching, and for regulating fertilization or development e.g., contraception and infertility.

Publications

  • Tseng, K., Y. Xie, J. Seeley, J.L. Hedrick, and C.B. Lebrilla. Profiling with structural elucidation of neutral and anionic O-linked oligosaccharide libraries by fourier transform mass spectrometry. Glycoconjugate Journal, 18, 309-320 (2001).
  • Barisone, G., J.L. Hedrick, and M. Cabada. The vitelline envelope of Bufo arenarum: biochemical and biological characterization. Biology of Reproduction, 66, 1203-1209 (2002).
  • Lindsay, L.L., J.C. Yang, and J.L. Hedrick. Identification and characterization of a unique Xenopus laevis egg envelope component, ZPD. Devel. Growth. Diff., 44, 205-212 (2002).
  • Vo, L.H., T.-Y. Yen, B. A. Macher, and J. L. Hedrick. Identification of the ZPC oligosaccharide ligand involved in sperm binding and the glycan structures of Xenopus laevis vitelline envelope glycoproteins. Submitted for publication (2002).


Progress 01/01/01 to 12/31/01

Outputs
Progress was made on methods development for sequencing biologically important oligosaccharides and cloning of egg envelope glycoproteins. 1) Exoglycosidase digestion in combination with the catalog library approach we developed earlier was used with matrix-assisted laser desorption/ionization Fourier transform mass spectrometry to obtain the complete structure of oligosaccharides derived from the Xenopus laevis egg jelly. Oligosacchride hydrolysis was used to independently confirm the validity of the catalog library approach. 2) To identify the jelly coat oligosaccharides that bind to the cortical granule lectin released at fertilization, the lectin was immobilized on the surface of a probe used in matrix-assisted laser desorption ionization mass spectrometry. This bioaffinity probe selectively bound oligosacchrides. Structural analysis of the bound oligiosaccharides identified that oliosaccharides with sulfate esters at the nonreducing ends were preferentially bound by the lectin. 3) The Xenopus laevis egg envelope is composed of six or more glycoproteins, three of which have been cloned and identified as mammalian homologs, ZPA, ZPB, and ZPC. The remaining glycoproteins are a triplet of high molecular weight components selectively hydrolyzed by the hatching enzyme. We have isolated one of these glycoproteins and cloned its cDNA. The mRNA for the protein was expressed only in early stage oocytes, as are other envelope components. From the deduced amino acid sequence, it was: 1) a secreted glycoprotein with a characteristic ZP domain in the C-terminal half of the molecule, 2) the N-terminal half was unrelated to any known glycoprotein 3) comparative sequence analysis of the ZP domain indicated that it was derived from an ancestor of ZPA and ZPB, with the greatest identity to ZPA. We designated this new envelope component ZPAX.

Impacts
The results of this research will improve our ability to determine the structure of biologically relevant oligosaccharides and understand their role in fertilization and early development functions. Knowledge of the macromolecules composing the egg envelope is potentially useful in understanding sperm-egg interactions and the process of embryo escape from the egg envelope (hatching). This knowledge can be used for regulating fertilization or development e.g., preventing fertilization when it is not wanted (contraception) or obtaining it when it is wanted (infertility).

Publications

  • Xie, Y., K. Tseng, J.L. Hedrick, and C.B. Lebrilla. Targeted use of exoglycosidase digestion for the structural elucidation of neutral O-linked oligosaccharides. Journal of the American Society for Mass Spectrometry 12, 877-884 (2001).
  • Lindsay,L.L., M.A. Wallace, and J.L. Hedrick. A hatching enzyme substrate in the Xenopus laevis egg envelope is a high molecular weight ZPA homolog. Dev. Growth. & Diff. 43, 305-313 (2001).
  • Tseng K., H. Wang, C.B. Lebrilla, B. Bonnell, and J.L.Hedrick. Identification and structural elucidation of lectin-binding oligosaccharides by bioaffinity matrix-assisted laser desorption/ionization Fourier transform mass spectrometry. Anal Chem. 73, 3556-61 (2001).


Progress 01/01/00 to 12/31/00

Outputs
Progress was made on several aspects of sperm-egg interactions. In addition to binding to the egg envelope, egg lectins in salmon and trout were shown to inhibit the motility of and to agglutinate sperm. Lectin-sperm interactions may contribute to a block to polyspermy in fish. An acrosin-like protease was detected in the head of the Xenopus sperm using antibodies against boar sperm acrosin. This sperm enzyme may play a role in envelope penetration. Binding of Xenopus sperm to the egg envelope involved predominantly the glycoprotein ZPC as the ligand. The oligosaccharide moiety was the critically important component of ZPC for binding. The cortical granule lectin present in Xenopus eggs was shown to exist in several mammalian species (including human) by cDNA cloning and immunological experiments. Immunocytochemical localization studies demonstrated its presence in the cortical granules before egg activation, and in the zona pellucida after activation in mouse and pig eggs. Thus, the lectin-ligand block to polyspermy hypothesis developed using Xenopus is relevant to mammals. The lectin-ligand interaction also provides a regulatory mechanism for protecting the developing embryo from environmental factors, both chemical and microbiological.

Impacts
These studies will contribute to our understanding of the basic mechanisms of the fertilization process. This information can potentially be used to regulate the fertilization process and enhance reproductive efficiency in agriculturally important animals.

Publications

  • Lindsay, L.L., and J.L. Hedrick. Immunolabeling and characterization of an acrosin-like protease in Xenopus laevis sperm. Molecular Biology of the Cell 11, 521a (2000).
  • McDougall, K., J.L. Hedrick, and B.D. Bavister, In situ pH measurements of the Syrian hamster uterus during early pregnancy to determine the role of pH in zona pellucida loss in vivo, Reproduction, Fertility,and Development 12, 105-111 (2000).
  • Murata, K., S. Yasumasu, Y. M. Lee, and J. L. Hedrick. Fish egg lectins: Important factors for a polyspermy block during fertilization. Molecular Biology of the Cell 11, 405a (2000).
  • Peavy, T.R., and J.L. Hedrick. An egg cortical granule lectin and the block to polyspermy in mammalian eggs. Molecular Biology of the Cell 11, 405a (2000).
  • Vo, L.H., and J.L. Hedrick. Independent and hetero-oliogmeric dependent sperm binding to egg envelope glycoprotein ZPC in Xenopus laevis. Biology of Reproduction 62, 766-774 (2000).


Progress 01/01/99 to 12/31/99

Outputs
Progress was made characterizing the egg envelope ligand for sperm binding, cloning egg proteases released upon egg activation, and clonging an additional egg envelope glycoprotein. A sperm binding assay was developed and used to identify ZPC as the envelope ligand. N-linked oligosaccharides with terminal N-acetylglucosamine residues were the functional moiety of ZPC. CDNA for the protease ovochymase was cloned revealing the presence of a polyprotein with three protease domains and five CUB domains similar to that found in oviductin and the hatching enzyme. A new egg envelope component, involved in embryonic hatching was cloned and term ZPAX because of its similarity with ZPA.

Impacts
These studies will contribute to our understanding of the basic mechanisms of the fertilization process. This information can potentially be used to regulate the fertilization process and enhance reproductive efficiency in agriculturally important animals.

Publications

  • Mozingo, N.M. and Hedrick, J.L. 1999. Distribution of lectin binding sites in Xenopus laevis egg jelly. Develop. Biol. 210:428-439.
  • Lindsay, L.L., Wieduwelt, M.J. and Hedrick, J.L. 1999. Oviductin, the Xenopus laevis oviductal protease that processes egg envelope glycoprotein gp43, increases, sperm binding to envelopes, and is translated as part of an unusual mosaic protein composed of two protease and several CUB domains. Biol. Reprod. 60:989-995.
  • Bonnell, B.S., Smith, S. G. and Hedrick, J.L. 1999. Dichromatic staining of electrophoretically separated extracellular matrix macromolecules. Analytical Biochem. 271:91-93.
  • Tseng, K., Hedrick, J.L. and Lebrilla, C.B. 1999. Catalog-library approach for the rapid and sensitive structural elucidation of oligosaccharides. Analytical Chem. 71:3747-3754.
  • Lindsay, L.L., Yang, J.C. and Hedrick, J.L. 1999. Ovochymase, an Xenopus laevis egg extracellular protease, is translated as part of a polyprotein. Proc. Natl. Acad. Sci. (USA) 96:11253-11258.
  • Vo, L.H. and Hedrick, J.L. 2000. Independent and hetero-oliogmeric dependent sperm binding to egg envelope glycoprotein ZPC in Xenopus laevis. Biol. Reprod. 62:766-774.


Progress 01/01/98 to 12/01/98

Outputs
Progress was made characterizing the extracellular matrix ligand for the cortical granule lectin, cloning egg proteases released upon egg activation, and identification of an egg envelope glycoprotein. The oligosaccharides of the ligand were released and characterized using matrix-assisted laser desorption fourier transform mass spectrometry. A structural pattern on non-reducing terminal sulfation was observed associated with ligand activity. cDNA for the protease ovochymase was cloned revealing the presence of a polyprotein with three protease domains and five CUB domains. The CUB domains may play a role in binding the protease to the egg extracellular matrix. The salmon egg lectin was completely sequenced and its envelope associated ligand was isolated.

Impacts
(N/A)

Publications

  • LINDSAY, L.L., and HEDRICK, J.L. 1998. Treatment of Xenopus laevis coelomic eggs with trypsin mimics pars recta oviductal transit by selectively hydrolyzing envelope glycoprotein gp43, increasing sperm binding to the envelope and rendering.
  • MORI, E., HEDRICK, J.L., WARDRIP, N.J., MORI, T., TAKASAKI, S. 1998. Occurrence of reducing terminal N-acetylglucosamine 3-sulfate and fucosylated outer chains in acidic N-glycans of porcine zona
  • LINDSAY, L.L., and HEDRICK, J.L. 1998. Extracellular matrix proteases of the Xenopus egg are translated together as a polyprotein. Molecular Biology of the Cell. 9, Supplement: 368.
  • PEAVY, T.R., CARROLL, E.J., Jr. and HEDRICK, J.L. 1998. Evolution of the ZPC gene family in amphibians. Molecular Biology of the Cell. 9,
  • BONNELL, B.S., TSENG, K., VO, L.H., LEBRILLA, C.B., and HEDRICK, J.L. 1998. Carbohydrate analysis of Xenopus laevis cortical granule lectin egg jelly coat ligand. Molecular Biology of the Cell. 9, Supplement:
  • VO, L.H., and HEDRICK, J.L. 1998. Sperm binding to the ZPC component of egg envelopes in Xenopus laevis. Molecular Biology of the Cell. 9, Supplement: 1803.
  • YASUMASU, S. and HEDRICK, J.L. 1998. Fish egg lectin and its ligand. Molecular Biology of the Cell. 9, Supplement: 1807.


Progress 01/01/97 to 12/01/97

Outputs
Progress was made characterizing the extracellular matrix ligand for the cortical granule lectin, characterizing proteases released upon egg activation, and cloning of an egg envelope glycoprotein. The oligosaccharides of the ligand were released and characterization initiated using matrix-assisted laser desorption fourier transform mass spectrometry initiated. The protease ovochymase was partially cDNA sequenced revealing the presence of protease domains and several CUB domains. The CUB domains may play a role in binding the protease to the egg extracellular matrix. The envelope glycoprotein gp43 was cloned and shown to be homologous to mammalian and fish egg envelope glycoproteins.

Impacts
(N/A)

Publications

  • BONNELL, B.S., and HEDRICK, J.L. 1997. Xenopus laevis cortical granule lectin/ligand interactions and their involvement in the block to polyspermy. J. Reproduction and Development 43:153-154.
  • YANG, J.C., and HEDRICK, J.L. 1997. cDNA Cloning and sequence analysis of the Xenopus laevis egg envelope glycoprotein gp43 (ZPC). Develop, Growth & Diff. 39:457-467.
  • LINDSAY, L.L., and HEDRICK, J.L. 1997. Proteases released from the activated or fertilized Xenopus laevis egg. J. Reproduction and
  • TSENG, K., LINDSAY, L.L., PENN, S., HEDRICK, J.L, and LEBRILLA, C.B. 1997. Characterization of neutral oligosaccharide-alditols from Xenopus laevis egg jelly coats by matrix-assisted laser desorption fourier transform mass spectrometry.


Progress 01/01/96 to 12/30/96

Outputs
Progress was made in three areas. 1. The oviductal protease, oviductin, which converts the unfertilizable coelomic envelope to the fertilizable vitelline envelope was cDNA cloned and characterized. The gene transcript consisted of a multidomain protein, possessing active and inactive protease domains and CUB domains. CUB domains are present in other developmentally regulated proteins. The may function in binding the oviductin to the egg envelope, thereby assisting in the specific, and limited hydrolysis of oviductin's substrate, the envelope glycoprotein ZPC. The function of the inactive protease domain is unknown. 2. The structures of the glycan moieties of the egg cortical granule lectin were determined. Two types of N-linked glycans were found, high mannose (Man8 and 9) and complex. The complex structures were tetra-antennary with nonreducing residues of 5-acetyl neuraminic acid, 4,5-diacetyl neuraminic acid, and N-acetyl galactosamine. The latter two monosaccharides are most unusual and likely responsible for the carbohydrate specific immunogenicity of the glycoproteins. 3. The ligand for the cortical granule lectin was isolated and characterized. Two high molecular weight glycoproteins (MWapp. of 630K and 450K) were visualized by electron microscopy and observed to be flexible rod-like molecules. The lectin was spherical shaped, and when added to the ligand, gave a complex appearing as "beads on a string.

Impacts
(N/A)

Publications

  • HEDRICK, J.L. 1996. Comparative structural and antigenic properties of zona pellucida glycoproteins. J. Reproduction and Fertility Suppl. 50:9-17 (1996).
  • QUILL, T. A., and HEDRICK, J.L. 1996. The fertilization layer mediated block to polyspermy in Xenopus laevis: isolation of the cortical granule lectin ligand. Archives of Biochemistry and Biophysics 333:326-332 (1996).
  • MOZINGO, N. M., and HEDRICK, J.L. 1996. Localization of the cortical granule lectin ligand in Xenopus laevis eggs. Development Growth & Differentiation 38:647-652.
  • BONNELL, B. S., and HEDRICK, J.L. 1996. Purification and analysis of XENOPUS LAEVIS cortical granule lectin and its ligand. Molecular Biology of the Cell 7:481a.
  • LINDSAY, L. L., and HEDRICK, J.L. 1996. Characterization of proteases released during activation of the XENOPUS LAEVIS egg. Molecular Biology of the Cell 7:482a.


Progress 01/01/95 to 12/30/95

Outputs
Progress was made on the vitelline (VE) to fertilization envelope (FE) conversion. The egg protease, ovochymase, was purified, cloned and sequenced. It is a member of the serine active protease II family with greatest identity to elastase. It is localized in the perivitelline space rather than in the egg cortical granules. It will not, however, convert the vitelline envelope (proteolysis of the 69K glycoprotein) by itself. Other proteases are involved in the VE to FE conversion process. In addition to a trypsin-like proteases identified in earlier studies, we found a prolyl endoprotease activity in the cortical granule exudate. We are currently investigating the role of this protease in the VE to FE conversion process. The proteases likely work in concert to convert the VE to the FE and effect a block to polyspermy. We cloned the envelope glycoprotein gp37. We established that it is homologous to the mammalian zona pellucida glycoprotein ZPB. Its amino acid sequence is 41.6% identical to that of human ZPB. The other frog egg envelope glycoproteins are also homologues of the mammalian zona pellucida glycoproteins (with human glycoproteins, ZPA is 28.5% and ZPC is 40.9% sequence identical). These findings of phylogenetic conservation of egg envelope glycoproteins establish the relevance of the frog model system to, mammals for studying the molecular mechanisms of fertilization.

Impacts
(N/A)

Publications

  • LINDSAY, L.E. and HEDRICK, J.L. 1995. Isolation and characterization of ovochymase, a chymotrypsin-like protease released during XENOPUS LAEVIS egg activation. Developmental Biology 167:513-515.
  • QUILL, T.A. and HEDRICK, J.L. 1994. Oviductal localization of the cortical granule lectin ligand involved in the block to polyspermy in XENOPUS LAEVIS. Develop. Growth & Differ. 36:615-620.


Progress 01/01/94 to 12/30/94

Outputs
During the last year, major progress was made on the vitelline to fertilization envelope conversion. The egg protease, ovochymase, responsible for limited proteolysis of the envelope glycoprotein gp69,64 was cloned. It is a serine active site enzyme that has sequence homology with chymotrypsin-like proteases. The gene for gp69,64 was previously cloned and we have ascertained that this glycoprotein has homology with the mammalian zona pellucida glycoprotein designated ZP2. This establishes that egg envelope glycoproteins are evolutionarily conserved between anurans (frogs) and mammals. We also cloned the gene for another envelope glycoprotein designated gp43. It was determined to have homology with the mammalian glycoprotein designated ZP3. The envelope glycoprotein gp43 is the substrate for the oviductal enzyme, oviductin, which converts the sperm impenetrable coelomic envelope to the sperm penetrable vitelline envelope. We have commenced cloning the gene for oviductin using a polymerase chain reaction strategy. We generated specific probes for oviductin and should complete the cloning for oviductin during the coming year.

Impacts
(N/A)

Publications

  • MOZINGO, N.M. and HEDRICK, J.L. 1994. Heterogeneous distribution of lectin binding sites in Xenopus laevis egg jelly. Mol. Biol. Cell 5:224a.
  • YANG, J.C., and HEDRICK, J.L. 1994. Cloning of gp69,64 and ovochymase from Xenopus laevis eggs. Mol. Biol. Cell 5:461a.


Progress 01/01/93 to 12/30/93

Outputs
During the last year, research on the coelomic to vitelline envelope conversion was temporarily halted due to a lack of personnel. The project was interrupted at the stage of cloning the gp43 envelope glycoprotein and the pars recta protease (oviductin) responsible for the conversion. Progress on the vitelline to fertilization envelope conversion includes: Isolation of the clone for the gp69, 64 envelope glycoprotein. The clone is apparently full length (includes a MET start site, leader sequence, stop site, and poly A tail but does not include the amino acid sequence of gp69,64 determined by Edman amino acid sequencing. We either have the wrong clone or an incorrect amino acid sequence. We are currently investigating both possibilities. We have also commenced cloning gp37, the envelope glycoprotein which presumably functions in sperm binding.

Impacts
(N/A)

Publications

  • TAKASAKI, S., MORI, E., HIRANO, T., FURUKAWA, K., AMANO, J., MORI, T., HEDRICK, J.L., WARDRIP, N.J. and KOBATA, A. 1993. Structure of Sugar Chains Included in Procine Zona Pellucida Glycoproteins. J. of Reproduction and Development, 39,.
  • HEDRICK, J.L. 1993. The Pig Zona Pellucida: Sperm Binding Ligands, Antigens, and Sequence Homologies. In: Reproductive Immunology. Eds. DONDERO, F. and JOHNSON, P.M., Serano Symposia Publications, Vol. 97, Raven Press, New York, pp. 59-65.
  • HEDRICK, J., CHANG, B., WARDRIP, N., QUILL, T. 1993. The Xenopus laevis Cortical Granule Lectin and Its Ligand. J. of Reproduction and Development, 39, Supplement: 81-82.
  • HIRANO, T., TAKASAKI, S., HEDRICK, J.L., WARDRIP, N.J., AMANO, J., and KOBATA, A. 1993. O-Linked neutral sugar chains of procine zona pellucida glycoproteins. European J. of Biochem. 214, 769.


Progress 01/01/92 to 12/30/92

Outputs
Progress during the last year on the coelomic to vitelline envelope conversion includes: preparation of a gammaZAP ovarian cDNA library, isolation of cDNA clones for the gp43 envelope glycoprotein and partial cDNA sequencing of relevant clones. A gammaZAP oviductal library was constructed and oligonucleotides prepared to the amino acid sequence of oviductin. The gp57 vitelline envelope component was proven to be pyruvate kinase derived from the egg; it is not a true envelope constituent and is not relevant to the fertilization process. Progress on the vitelline to fertilization envelope conversion includes: isolation of gp69, 64 clones from a gammaZAP ovarian cDNA library and partial cDNA sequencing of relevant clones. Oligonucleotide primers were prepared to amino acid sequences of ovochymase for use in a PCR approach to cloning the enzyme. The cloning of the cortical granule lectin (CGL) cDNA was completed. The CGL is structurally unique with no homologous proteins in the sequence database. The functionally important oligosaccharides of CGL have been isolated and partially characterized. High mannose (8 & 9) and complex glycans are present on two N-glycosylation consensus sequence sites. A highly immunogenic sialic acid derivative of unknown structure is present on the complex glycans.

Impacts
(N/A)

Publications

  • HARDY, D.M. and HEDRICK, J.L. 1992. Oviductin: Purification and properties of an oviductal glycoproteinase essential for XENOPUS LAEVIS fertilization. Biochemistry 31:4466-4472.
  • BIRR, C.A. and HEDRICK, J.L. 1992. Immunoelectrophoretic identification of jelly coat ligands bound by the cortical granule lectin from XENOPUS LAEVIS eggs. Dev., Growth and Differ. 34:91-98.
  • FABRY, H. and HEDRICK, J.L. 1992. Antibody production in the goat: immunokinetics and epitope specificity using a glycoprotein immunogen. Zool. Sci. 9:995-1000.
  • LINDSAY, L., LARABELL, C.A., and HEDRICK, J.L. 1992. Localization of a chymotrypsin-like protease to the perivitelline space of XENOPUS LAEVIS eggs. Dev. Biol. 154:433-436.